Transient molecular interactions between macromolecules provide a powerful mechanism in biology to regulate function and cell processes. A crucial step toward the full understanding of cellular systems consists of mapping the networks of physical DNA-, RNA- and protein-protein interactions of an organism of interest as completely and accurately as possible, the “interactome network.” Recently, a large number of biological pathway and network databases have been developed to capture the expanding knowledge of molecular interactions. However, the complete understanding of molecular interactions requires high-resolution 3D structures as they provide key atomic details about binding interfaces and information about structural changes that accompany molecular interactions.
The interactions of two or more dissimilar proteins, the so-called protein-protein interactions (PPIs), are central to most biological processes. Critical cellular functions, including cell growth, DNA replication, transcription activation, translation and transmembrane signal transduction, are all regulated by multiprotein complexes and, therefore, quaternary protein structures represent a large and attractive emerging class of targets for human therapeutics.1-4 It is now well established that human diseases can be caused by aberrant PPIs, either through the loss of an essential interaction or through the formation and/or stabilization of a protein-protein interaction at an inappropriate time or location. Proteins themselves are dynamic and can exist in multiple conformations, often induced by interaction with another protein in a (transient) protein-protein interaction. These conformational changes are often functionally important and reflect allosteric regulation that activate or inactivate specific protein functions. The diversity and complexity of these highly dynamic PPIs present many opportunities and challenges for the identification of drug-like molecules with the ability to modulate the PPI with the necessary selectivity and potency.
PPIs can occur between identical or non-identical chains (homo- or hetero-oligomers). Besides composition, non-obligate and obligate complexes can be distinguished. In an obligate PPI, the protomers are not found as stable structures on their own in vivo. The components of non-obligate complexes are independently stable. In contrast to a permanent interaction that only exists in its complexed form, a transient interaction associates and dissociates in vivo. Weak transient interactions that feature a dynamic oligomeric equilibrium in solution, where the interaction is broken and formed continuously and strong transient associations that require a molecular trigger to shift the oligomeric equilibrium are distinguished. Many PPIs do not fall into distinct types. Rather, there is a continuum between obligate and non-obligate interactions, and the stability of transient complexes varies much depending on physiological conditions and environment.5 
Yeast two-hybrid screens have been used extensively to map binary transient interactions and tandem affinity purification run in conjunction with mass spectroscopy and chemical cross-linking has been developed to detect transiently formed complexes. A large number of biological network databases have been developed to capture the expanding knowledge of protein-protein interactions but rigorous assessment of high-throughput as well as literature-curated PPI data has shown that experimental data can be prone to error and are not completely comprehensive.5 Therefore, computational methods can be applied to increase confidence and predict interactions currently hidden from the experimental techniques.6 
Ultimately, the complete understanding of molecular interactions requires high-resolution 3D structures as they provide key atomic details about binding interfaces and information about structural changes that accompany protein-protein interactions. The structural details of these interactions, often necessary to understand their function, are only available for a tiny fraction and this gap is growing.7 Modern overexpression and purification procedures can usually supply sufficient material for structural studies on a single protein, but obtaining sufficient material can be an enormous problem for large multiprotein complexes. But even if expression and purification problems can be overcome, we are still confronted with the intrinsic property that these complexes are transient, complicating their structural characterization X-ray crystallography, Nuclear Magnetic Resonance (NMR), Small Angle X-ray Scattering (SAXS) or Electron microscopy (EM). The transient nature of these PPIs also makes them difficult targets for drug discovery.
The structural characterization of multiprotein complexes is currently limited to permanent protein-protein interactions or to transient interactions that can be stabilized by i) binding of small molecule effectors (such as nucleotides, substrates, ions, or analogs thereof), ii) naturally occurring ligands, or iii) introduction of stabilizing mutations to the interacting protomers of the PPI. Antibodies and fragments derived thereof have been identified that are able to bind quaternary protein structures.8 However, none of these antibodies (or fragments) selectively stabilize the PPI, i.e., preferentially interact with the protein complex versus one of the interacting protomers. Camelid single-domain antibody fragments (VHHs or NANOBODIES®) have been identified that bind conformational epitopes of (membrane) proteins and complexes thereof (Pardon et al., 2014). For example, NANOBODIES® that stabilize a complex composed of agonist-occupied monomeric β2-adrenergic receptor and nucleotide-free Gs heterotrimer were identified by (i) immunizing llamas with the complex after chemical cross-linking of the associating proteins to mature NANOBODIES® that bind allosteric epitopes on the transient complex, and (ii) two different panning strategies. These NANOBODIES® were able to protect the complex from dissociation by GTPγS and provide stabilization to the G protein subunits, which was essential for determining its crystal structure. Conformational antibodies that stabilize particular conformers of single proteins and methods to identify these have also been described (Rasmussen et al., 2011; Kruse et al., 2013). However, generic methods to identify allosteric modulators that bind at a site orthogonal to the protein-protein interface, inducing conformational changes that affect the protomers' propensity to form a complex, are lacking.
There is, thus, a need for straightforward methods for the selection of novel tools that selectively stabilize transient protein complexes, making them amenable for structural investigation and drug discovery.